(1) Field of the Invention
The present invention relates to a novel cDNA encoding a novel polypeptide including a hevein sequence of 43 amino acids. In particular the present invention relates to a novel cDNA encoding a polypeptide of 204 amino acids.
(2) Prior Art
Latex of the rubber tree (Hevea brasiliensis) is produced by highly specialized cells known as laticifers (de Fay, E., et al., in Physiology of Rubber Tree Latex, eds. d'Auzac, J., Jacob, J. L., and Chrestin, H. (CRC, Boca Raton, Fla.), pp. 3-14 (1989)). The contiguous end walls of adjacent laticifer cells are perforated, thus forming an anastomosing system. Upon wounding, the common cytoplasmic content of these cells is expelled in the form of latex. Sealing of wound sites occurs by coagulation of the outflowing latex. This process involves bursting of the lutoid bodies (organelles of vacuolar origin) and subsequent interaction of the released cationic proteins with the negatively charged rubber particles (d'Auzac, J., et al., in Physiology of Rubber Tree Latex, eds. d'Auzac, J., et al., (CRC, Boca Raton, Fla.), pp. 59-88 (1989)). Wound plugging may be important in preventing entry of pathogens in the phloem.
One of the major proteins in the lutoid bodies of rubber tree latex is hevein (Archer, B. L., et al., J. Rubber Res. Inst. Malaya 21, 560-569 (1969)). Hevein is a small, single chain protein of 43 amino acids unusually rich in cysteine and glycine (Walujono, K., et al., in Proc. Internat. Rubber Conf. 518-531 (1975)). Recently, hevein has been shown to bind chitin and to inhibit the growth of several chitin-containing fungi Van Parijs, J., et al., Planta 183:258-264 (1991)). It has therefore been suggested that hevein plays a role in the protection of wound sites from fungal attack Van Parijs, J., et al., Planta 183:258-264 (1991)).
Various classes of chitin-binding proteins have been reported to contain polypeptide domains homologous to the hevein sequence. The lectins from the monocotyledonous species wheat, barley and rice are composed of four repetitive hevein-like domains (Wright, C. S., et al. Biochemistry 23, 280-287 (1984); Raikhel & Wilkins, Proc. Natl. Acad. Sci. U.S.A. 84, 6745-6749 (1988); Lerner, D. R., et al., Plant Physiol. 91, 124-129 (1989); and Wilkins, T. A., et al., Plant Cell 1, 541-549 (1989)), whereas a related lectin from the dicotyledonous Urtica dioica is thought to be composed of two such domains arranged in tandem (Chapot, M. P., et al., FEBS Lett. 195, 231-234 (1986)). Basic chitinases from bean (Broglie, K. E., et al., Proc. Natl. Acad. Sci. U.S.A. 83, 6820-6824 (1986)), tobacco (Shinshi, H., et al., Proc. Natl. Acad. Sci. U.S.A. 84, 89-93 (1987)) and poplar (Parsons, T. J., et al. Proc. Natl. Acad. Sci. U.S.A. 86, 7895-7899 (1989)) have a single hevein-like domain located at the amino-terminus which is fused to an unrelated carboxyl portion. Likewise, two wound-induced genes from potato encode proteins with a hevein-like domain located at the amino-terminus and a carboxyl-terminal extension that differs from the chitinase carboxyl-terminus (Stanford, A., et al., Mol. Gen. Genet. 215, 200-208 (1989)).
Hevein was first isolated from the latex of the rubber tree (Hevea brasiliensis) (Archer, B. L., et al, J. Rubber Res. Inst. Malaya 21, 560-569 (1969)). In the latex, hevein is present in lutoid bodies, which are small, vacuole-derived organelles. Hevein is a major protein in these particles representing up to 70% of the total protein content (Archer et al., previously cited). It is not known at present whether hevein occurs in other parts of the plant. Hevein is a monomer and has an apparent molecular weight of 9.5 kDa as determined by gel filtration and SDS-PAGE Van Parijs, J., et al., Planta 183:258-264 (1991)). The amino acid sequence of hevein has a high degree of homology to WGA (Wright, C. S., et al., Biochemistry 23, 280-287 (1984)). The antifungal effects of hevein was examined on three different test organisms (Trichoderma hamatum, Phycomyces blakesleeanus, and Botrytis cinerea) by the hyphal growth inhibition assay. Dose-response experiments were carried out for each combination of hevein and fungus to determine the hevein concentration required for 50% inhibition of hyphal extension. Hevein caused 50% growth inhibition of T. hamatum, P. blakesleeanus, and B. cinerea at concentrations as low as 90, 300, and 250 ug/ml, respectively. Compared to tobacco chitinase (NTC), hevein is about one order of magnitude less active with T. hamatum and P. blakesleeanus, but more potent for inhibition of B. cinerea. Other fungi used in antifungal assays are Septoria nodorum, Pyricularia oryzae, Fusarium culmorum and Fusarium oxysporum which show a similar inhibition of hyphal growth as is shown for B. cinerea. Other chitin-binding lectins, WGA, Chelidonium majus agglutinin (CMA), Datura stramonium agglutinin (DSA) and Solanum tuberosum agglutinin (STA), did not affect fungal growth at concentrations below 2 mg/ml. To provide that the growth inhibiting effect of hevein was not due to contamination by chitinases or other compounds, this protein was subjected to several inhibitory treatments. The antifungal activity of hevein, and tobacco chitinase were reversed by addition of the specific ligand chitotriose (2 mM). However, hevein retained its antifungal properties when heated for 10 minutes at 90.degree. C., whereas chitinase activity was completely destroyed by the same treatment. Therefore, this small-sized chitin-binding protein, hevein has antifungal properties and represents a promising target gene for possible applications in genetic engineering experiments.
The hevein protein represents a promising target gene for genetic engineering experiments. First, this protein has been shown to be a potent inhibitor of fungal growth, and may thus contribute to the plant's own resistance to fungal infection. On a per gram basis, its antifungal potency is comparable to that of phytoalexins, which inhibit fungal growth in the concentration range of 50 to 100 ug/ml (Weinstein, L. I., et al., Plant Physiol., 68, 358-363 (1981)).